Mild traumatic brain injury (mTBI) has been shown to negatively alter bacterial diversity and composition within the gut, known as dysbiosis, in rodents and humans. These changes cause secondary consequences systemically through decreased bacterial metabolites such as short chain fatty acids (SCFAs) which play a role in inflammation and metabolism. The goal of the study was to identify if giving prebiotic inulin prior to closed head injury (CHI) could mitigate gut dysbiosis, increase SCFAs, and improve recovery outcomes, including protecting cerebral blood flow (CBF) and white matter integrity (WMI) in young mice.We fed mice at 2 months of age with either inulin or control diet (with cellulose as fiber source) for two months before the CHI and continued till the end of the study. We analyzed gut microbiome composition and diversity, determined SCFAs levels, and measured CBF and WMI using MRI. We compared the results with Naïve and Sham-injury mice at 24 hours, 1.5 months, and 3-4 months post-injury.We found that both CHI and Sham mice had time-dependent changes in gut composition and diversity after surgery. Inulin significantly reduced the abundance of pathobiont bacteria, such as E. coli, Desulfovibrio spp and Pseudomonas aeruginosa, in Sham and CHI mice compared to mice fed with control diet. On the other hand, inulin increased SCFAs-producing bacteria, such as Bifidobacterium spp and Lactobacillus spp, increased levels of SCFAs, including butyrate and propionate, and significantly altered beta diversity as early as 24 hours post-injury, which lasted up to 3-4 months post-injury. The mitigation of dysbiosis is associated with protection of WMI in fimbria, internal and external capsule, and CBF in the right hippocampus of CHI mice, suggesting protection of memory and cognitive functions.The results indicate that giving inulin prior to CHI could promote recovery outcome through gut microbiome modulation. As inulin, microbiome analysis, and MRI are readily to be used in humans, the findings from the study may pave a way for a cost-effective, accessible intervention for those at risk of sustaining a head injury, such as military personnel or athletes in contact sports. Consideprior to injury would have a better protection for gut microbiome and brain physiology, especially having a positive impact on CBF and WMI. Therefore, the goal of the study was to fill the knowledge gap. The outcomes from the study may provide useful and important information to better protecting brain functions for those who are at high risk for TBI, such as military personnel and athletes in contact sports.Emerging evidence shows that manipulation of the gut microbiome through dietary intervention may be actionable in reducing long-term symptoms after mTBI . In a re22.1via power analysis to ensure comparison with a = 0.05 and b = 0.20 for detecting a true difference of each measured variable between the groups. The Control groups were fed with a diet containing cellulose that is nonfermentable . The inulin diet was also from TestDiet . ad libitum access to the diets, and we measured the weight of the mice and the weight of the remaining food biweekly to estimate the food intake during the feeding period.Male C57BL/6 wild-type mice were used in the study. They were randomly assigned to one of the following six groups: Naïve-Control (n =11), Sham-Control (n = 12), CHI-Control (n = 12), Naïve-Inulin (n = 12), Sham-Inulin (n = 12) and CHI-Inulin (n = 12). The number of animals per group was determined 2.2Briefly, as previously described , mice we2.3™ DNA Mini Kit per manufacturers guidelines. According to the manufacturer’s instructions, genomic DNA (20 ng input) was used as template for shotgun metagenome library preparation using the Illumina DNA Prep (formerly Nextera FLEX) protocol. DNA was subject to tagmentation and then amplified using 10 cycles of PCR to incorporate unique dual indices. The amplified products were cleaned, and size selected by two incubations with SPB at 0.53X and 0.12X and eluted in water. Libraries were pooled and initially sequenced on an Illumina MiniSeq mid-output flowcell as part of library QC. Based on the output from the MiniSeq run, samples were re-pooled to produce even sequence yields. The final pool was sequenced on an Illumina NovaSeq6000 instrument using an S4 flow cell and paired-end 2×150 sequencing reads. Library preparation and QC sequencing were performed at the Genomics and Microbiome Core Facility (GMCF) at Rush University. NovaSeq sequencing was performed at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign (UIUC). The gene amplicon sequence data generated as part of this study have been submitted to the NCBI BioProject database (PRJNA903182).Fecal samples were collected in a two-hour window between 7-9 am. Samples were frozen at −80°C until DNA extraction. Genomic DNA was extracted from fecal samples and ZymoBIOMICSFor taxonomic annotation, raw reads were mapped to the NCBI nucleotide database using Centrifuge . TaxonomDifferential analyses of bacterial taxa as compared with experimental covariates  were performed using the software package edgeR on raw sequence count . Prior t2.42.4.1Post-hoc, pairwise tests were performed using Mann-Whitney test. Plots were generated in R using the ggplot2 library . Diffusion tensor imaging (DTI) is used to characterize microstructural changes in the brain  values were analyzed using DSI studio  at the Magnetic Resonance Imaging & Spectroscopy Center of UK. Mice were anesthetized with 4.0% isoflurane for induction and then maintained in a 1.2% isoflurane and air mixture using a facemask. Heart rate (90–130 bpm.), respiration rate (70-90 breaths per minute), and rectal temperature (37 ± 0.5°C) were monitored. A water bath with circulating water at 45–50°C was used to maintain the body temperature. Qualitative CBF was measured using arterial spin labeling method (Fair Rare perfusion weighted sequence) in an interleaved fashion (TI loop outside) with the following parameters: FOV = 20 x 20 mmhe brain . The imaI studio .2.6Samples were prepared for LCMS by adding 200 μL of isopropyl alcohol containing an internal standard 2-isobutoxyacetic acid (15 μg/mL) to 20 mg of cecal contents in a 1.5 mL microcentrifuge tube to reach a mass to volume ratio of 0.1 mg/μL. Samples were shaken for 30 min and then centrifuged for 30 min at 13,000×g. 50 μl of the supernatant was transferred to a glass sample vial insert and derivatized according to previously published method with some modifications . Briefly2.7All statistical analyses were completed using R 3.6.2 and GraphPad Prism 9.0 . Two-way ANOVAs were performed for determination of differences between groups. Based on the a-priori hypothesis constructed from our pilot study data, we expected a specific pattern of responses, leading us to plan a set of pairwise comparisons  to assess dietary effects. These planned comparisons were reported in addition to ANOVA results . Planned33.1Sham-control mice had a lower alpha diversity at 24 hours post-injury compared to the Naïve and CHI mice fed with control diet. The difference was not found in the shame-inulin mice . Alpha dBeta diversity (Bray-Curtis index) showed a time-dependent change, with increased diversity over time. It showed an overall effect due to diet at 24 hours post-injury  Sum of , there w3.2Escherichia coli (E. coli) and Shigella spp were significantly increased in Sham-Control mice  and CHI-Control mice . Interestingly, the Sham-Control mice had significantly higher magnitude of the increase in E. coli compared with the CHI-Control mice.We analyzed the microbiome composition across the three time points. We found that there was a time-dependent changes on microbiome composition among the Naïve, Sham and CHI mice. At 24 hours post-injury , EscheriDesulfovibrio spp and Lachnospiraceae bacterium 28-4 were significantly higher among all the three Control groups . The Naive and CHI mice had additional higher levels of Pseudomonas aeruginosa . At 3 months post-injury .At 1.5 months post-injury , we fount-injury , CHI-ConInterestingly, inulin was able to reduce the levels of the all pathobiont bacteria throughout the three time-points , A–C. InBifidobacterium spp in Naïve (B. other Q = 4.19e-04), Sham  and CHI  and higher levels of Lactobacillus spp in CHI mice (L. gasseri Q = 5.14e-03).At 24 hours post-injury , inulin Bifidobacterium spp in Naïve , Sham  and CHI  and higher levels of Lactobacillus spp. in Sham  and CHI .At 1.5 months post-injury , inulin Bifidobacterium spp. in Naïve , Sham  and CHI  and higher levels of Lactobacillus spp in Sham (L. johnsonii Q = 0.03) and CHI (L. gasseri Q = 3.75e-03).At 3 months post-injury , inulin Collectively, the data shows that there was a time-dependent changes in microbiome composition between the Naïve, Sham and CHI mice, but inulin was able to reduce the abundance of pathobiont bacteria and increase the abundance of beneficial bacteria.3.3All three SCFAs showed a significant effect due to diet three months post-injury. There were decreased levels of acetate  in inuli3.43.5CBF is shown as perfusion-weighted values with an arbitrary unit (A.U.). Pairwise comparisons in the right hippocampus showed a significant increase in CBF in the CHI-inulin compared to the CHI-control in the right hippocampus (p = 0.0390) . This is4E. coli at the first 24 hours after injury and then normalized , which is known to cause various life-threatening infections and alter intestinal barrier function  microbiota at as early as 24 hours post-injury. In particular, we found increased level of roducers . This isualities , and Lacathogens .Streptococcus species. It could also be due to interconversion of SCFAs from acetate to butyrate, which occurs at a high level in the cecum (Interestingly, we found that acetate was lower in the inulin-fed groups compared with controls. This could be because there are higher acetate-producing bacteria in the control-fed mice, such as commensal he cecum . Furtherhe cecum , which iInulin increased FA of the CHI mice in the right fimbria and right external capsule at 1.5 months post-injury, and in the right internal capsule at 3-4 months post-injury. The fimbria is the major white matter output tract of the hippocampus, where damage can cause difficulty in long term recall . The extWMI was not impacted in the Sham mice, but we observed that inulin enhanced WMI of Sham mice at 3-4 months post injury. Sham-inulin mice had higher FA in both sides of corpus callosum, and the left external capsule. The corpus callosum integrates and transfers information from both cerebral hemispheres to process sensory, motor, and cognitive signals . TraumatSimilar to the FA results, we found that inulin also protected CBF in the hippocampus of the CHI mice, suggesting a protection of cognitive function. This may also be associated with the changes in SCFAs, as butyrate is known to increase neurogenesis and reduce inflammation, which could be beneficial in terms of CBF and brain vascular integrity .APOE4 alleles, the strongest genetic risk factor for AD (Our findings are in line with another study showing that young male mice fed with various supplements of fruit or vegetable blends prior to moderate TBI, had reduced neuroinflammation and improved functional recovery compared to controls . These fr for AD .A limitation of this study is that only male mice were used. Female mice will be included in future studies to understand sex effects on injury and diet. It will also be critical to determine the underlying mechanism of CBF and WMI protection with inulin, and whether SCFAs play a key role. In this study, we reported SCFAs from the cecal content; plasma and brain SCFAs levels will also needed to be included in the future studies. Further, although we believe the transient dysbiosis in Sham mice comes primarily from anesthetic inhalation, a study that further elucidates the changes seen in Sham mice needs to be conducted. Another limitation is that the mice were singly housed in the study, which may induce stress. A recent study showed that there were no differences in exploratory activity, anxiety, working memory and fear memory processing in male C57BL/6JRj mice that were singly housed, pair housed or pair housed with a cage divider . HoweverIn summary, we found that inulin supplementation prior to injury may beneficially affect the gut microbiome by reducing pathobiont bacteria, increasing putatively beneficial bacteria, increasing SCFAs levels in cecal contents, and protecting WMI and CBF in CHI mice. As inulin, microbiome analysis, and MRI are readily to be used in humans, giving prebiotic fiber could be a cost-effective, accessible intervention for those at risk of sustaining a head injury, such as military personnel or athletes in contact sports.


Background and Objectives: Acute abdominal pain in children has been noticed to be a primary reason to seek medical attention in multisystem inflammatory disorder (MIS-C), which can prevail separately or together with acute appendicitis. Our aim was to distinguish regular appendicitis cases from MIS-C and to suggest the best clinical and laboratory criteria for it. Materials and methods: Cases of patients, admitted to the Pediatric Surgery Department over a six-month period in 2021, were retrospectively analyzed. Confirmed MIS-C or acute appendicitis cases were selected. MIS-C cases were either separate/with no found inflammation in the appendix or together with acute appendicitis. Acute appendicitis cases were either regular cases or with a positive COVID-19 test. Four groups were formed and compared: A-acute appendicitis, B-MIS-C with acute appendicitis, C-MIS-C only and D-acute appendicitis with COVID-19. Results: A total of 76 cases were overall analyzed: A-36, B-6, C-29 and D-5. The most significant differences were found in duration of disease A—1.4 days, B—4.5 days, C—4 days, D—4 days (p < 0.0001), C reactive protein (CRP) values A-19.3 mg/L B-112.5 m/L, C-143.8 mg/L and D-141 mg/L (p < 0.0001), presence of febrile fever A-13.9%, B-66.7%, C-96.6% and D-40% (p < 0.0001) and other system involvement: A 0%, B 100%, C 100% and D 20%. A combination of these factors was entered into a ROC curve and was found to have a possibility to predict MIS-C in our analyzed cases (with or without acute appendicitis) with an AUC = 0.983, p < 0.0001, sensitivity of 94.3% and specificity of 92.7% when at least three criteria were met. Conclusions: MIS-C could be suspected even when clinical data and performed tests suggest acute appendicitis especially when at least three out of four signs are present: CRP > 55.8 mg, symptoms last 3 days or longer, febrile fever is present, and any kind of other system involvement is noticed, especially with a known prior recent COVID-19 contact, infection or a positive COVID-19 antibody IgG test. The COVID-19 pandemic has been a difficult challenge to face globally with a lot of severe and fatal cases in adults. Children, however, seem to have been affected a lot less, making up less than 10 percent of all laboratory-confirmed cases, but with a possible untested higher prevalence, which was overlooked due to many children having milder symptoms, being asymptomatic or not being tested at all ,3. ChildOur aim was to compare and distinguish regular appendicitis cases from MIS-C and suggest the best clinical and laboratory criteria for it, as well as to see if COVID-19-positive appendicitis cases resemble MIS-C or are just regular acute appendicitis cases.We retrospectively analyzed cases of patients, who were hospitalized with abdominal pain to the Pediatric Surgery Department at the Lithuanian University of Health Sciences Hospital over a six-month period in 2021 as well as selected cases with operated acute appendicitis or multisystem inflammatory disorder. MIS-C cases were either found to be separate  or with confirmed acute appendicitis . Regular acute appendicitis cases were found to be either separate or had a positive COVID-19 PCR Antigen test (without MIS-C criteria). The cases found were separated into the four following groups: Group A—patients with only acute appendicitis; group B—patients with acute appendicitis, who were also confirmed to have MIS-C; group C—patients with MIS-C without acute appendicitis; and group D—patients with acute appendicitis, who were found to be COVID-19 PCR Antigen positive . Surgically treated patients that had minimal or no inflammatory changes in the appendix and were diagnosed with MIS-C were categorized into group C.We retrospectively analyzed their medical records and compared their demographic data , duration of symptoms (in days), typical appendicitis symptoms, as well as MIS-C typical symptoms , laboratory tests on admission (C Reactive Protein (CRP), white blood cell (WBC) count, neutrophil percentage, platelet count and hemoglobin), ultrasound data as well as the pediatric appendicitis score (PAS) were counted for each patient. CT data was only interpreted in terms of respiratory system damage as a chest and abdominal CT was only performed for one patient in group B.The stage of inflammation was taken from the histopathology report if the appendix was removed. Phlegmonous appendicitis was categorized as uncomplicated, while gangrenous or perforated was categorized as complicated. If surgical drainage due to an abscess or infiltrate was performed without removing the appendix (due to inability to remove it because of surrounding tissue damage), the stage of appendicitis was categorized as complicated. If an operation was performed but the appendix was found with minimal changes, which was confirmed on pathology report, it was not counted as acute appendicitis. A prior known positive test for COVID-19 PCR Antigen (in the last two months) was noted, as well as PCR COVID-19 antigen test results upon hospitalization or later until discharge and COVID-19 antibody IgG test results when the test was performed.MIS-C was confirmed according to the WHO criteria: children/adolescents, with fever ≥3 days, at least two organ system involvement and evidence of COVID-19 or likely contact with COVID-19 infected patients , as decip < 0.05. Significant symptoms were added into a score, which was tested out using a receiver-operated characteristic curve (ROC), with a significant area under curve (AUC) > 0.5 when p < 0.05 was counted as significant and sensitivity/specificity was measured for the test.Statistical data analysis was performed by using IBM SPSS Statistics software. Quantitative data was found not to have a normal distribution using Kolmogorov and Smirnov tests, so a nonparametric Kruskal–Wallis test was used for the analysis. Results are described as median . Qualitative data was compared using a Chi square for homogeneity criterion. Results are presented in absolute numbers and percentages. Obtained differences and relations were found significant if p = 0.02. Symptoms lasted significantly less in group A than in all others, with a median of 1.4  days vs. 4.5 days (group B) vs. 4 days (group C) vs. 4 days (group D), p < 0.0001.A total of 76 cases were overall analyzed for the study: 36 cases of operated acute appendicitis—group A; 6 operated acute appendicitis cases which later were confirmed to have MIS-C—group B; 29 cases with MIS-C —group C; and 5 patients surgically treated for acute appendicitis with a confirmed COVID-19 infection —group D. We found that gender distribution was insignificant  and rebound tenderness (55% and 60%), while the MIS-C groups did not , p < 0.0001. Both MIS-C groups showed 100% of other system involvement , while acute appendicitis cases showed no other system involvement (but were not tested for specific disorders if they did not exhibit any symptoms), and group D had a proven coagulation disorder in one patient, who was tested; however, others did not have any symptoms and thus did not receive additional testing.The most common symptom overall was right lower quadrant pain, which had a high incidence in group A (86.1%), group B (100%) and group D (100%) but was mostly absent in group C (31%), p < 0.0001) and had the lowest median diameter of the appendix 0.6 cm . Lymph node hyperplasia was present in a third of group B patients (33.3%) and group C patients (35.7%), while it was present in almost no group A or D patients (p = 0.011). Free fluid was not present in group B (0%) but was noticed in about half of group A patients (44.4%) and a little more than a half of group C patients (64.3%) and group D patients (60%), p = 0.03.Abdominal ultrasound analysis showed that the appendix was significantly the least visualized in solitary MIS-C cases (group C) in only 28.6%  × 109/L) and platelet ) counts were observed in MIS-C (group C) p = 0.004 and p < 0.0001, while the highest hemoglobin levels were found in the acute appendicitis group A—Hgb 136  g/dL, p = 0.0001, but neutrophil percentage did not differ between the groups.Laboratory tests showed that only group D cases had a positive PCR Antigen test, while 16.7% of cases in group B had a positive test prior to admission and 27.6% of cases in group C. All patients in both MIS-C groups who were tested for COVID-19 Antibody IgG had positive results. A very distinguishable and significant difference was noted in CRP levels: group A had the lowest median ) in comparison to other groups , group C 143.8 mg/L  and group D 141 mg/L ), p < 0.0001, a sensitivity of 91.4% and specificity of 82.9% when it is more than 55.8 mg/L.Upon analyzing these factors, we wanted to analyze if CRP alone and in combination with other factors could help to distinguish MIS-C (both with and without acute appendicitis) cases from acute appendicitis or acute appendicitis with COVID-19 in our data. We analyzed area under curve for C reactive protein to distinguish MIS-C cases from others . We founThis has prompted us to see if a combination of CRP and other significant factors could improve these results. Upon closer inspection of previously analyzed data, we combined these factors into an MIS-C score .p < 0.0001 and a sensitivity of 94.3%, a specificity of 92.7% when at least three criteria were met.We applied this calculated score to our data in The importance of viruses in the pathogenesis of acute appendicitis has not yet been proven for certain. The possibility exists that viruses have influence either directly or indirectly, as cases where acute appendicitis was diagnosed with the presence of a virus have been presented in the literature. The viruses identified and mentioned are the Cytomegalovirus, Ebstein Barr virus, Rota virus, Adenovirus, Influenza virus, Varicella Zoster and even the Dengue virus ,20,21,22The rise of COVID-19 with extensive testing has presented us with more possibilities than ever before to study the importance of a virus in the development of acute appendicitis. Although children make up the minority of laboratory confirmed cases with milder symptoms, this could be contributed to having a good early innate immune response to clear the virus ,7,14. ItThe link to acute appendicitis has been put up for debate due to a very prominent gastrointestinal presentation in MIS-C . AlthougWe have found both cases of MIS-C presenting with acute appendicitis, which required surgical treatment, as well as MIS-C cases where conservative treatment was sufficient. In our patients, we found that 17% (6/35) of all MIS-C cases in the year of 2021 were confirmed cases of acute appendicitis, of which 66.6% were complicated. On the other hand, we also had COVID-19 positive cases with acute appendicitis, with an 80% complicated appendicitis rate. High rates of complicated appendicitis could be linked to similar cytokine patterns discussed above.We did not find any significant gender predominance; however, while acute appendicitis patients and MIS-C with appendicitis had a comparable median age, COVID-19 positive patients tended to be older, with a median of 13 years, and MIS-C patients without appendicitis were younger, with a median of 8 years. A very notable difference was found in terms of duration of symptoms: acute appendicitis cases had a median of 1.4 days duration and both MIS-C groups as well as the COVID-19 group had a much longer duration of symptoms of about 4 days. The long duration of symptoms could be explained by some patients seeking treatment earlier and having been prescribed antibiotics without referral to the hospital, but only ended up hospitalized and treated when the abdominal pain was more prominent, and the intensity and amount of symptoms increased with time. The need for intensive care in all MIS-C cases was about 45.7%, which confirms the numbers presented in the literature [While regular acute appendicitis cases did not exhibit other system involvement, the same could be said about COVID-19 appendicitis cases, as MIS-C cases both with and without acute appendicitis show a high presence of cardiovascular involvement as mentioned in the literature . We founUltrasound has been described as unable to help differentiate between acute appendicitis and MIS-C due to a thickening of the appendix along with the distal ileum . We foun9/L (vs. 14.1 × 109/L in acute appendicitis), but MIS-C cases without appendicitis had a normal white blood cell count 8.4 × 109/L. C reactive protein levels were found high (>100 mg/L) in all patient groups except regular acute appendicitis cases, which could be explained by an intense inflammatory response, multiple organ system involvement in MIS-C, as well as simply a longer duration of symptoms—as CRP is not only inflammation degree, but very time dependent [12/L. These results are comparable to the literature, as thrombocytopenia and low WBC count have been used to differentiate from acute appendicitis, even with appendiceal lumen increase on ultrasound [Typical laboratory tests also had interesting outcomes. While Neutrophilia was present in different degrees and comparable in all groups, confirming other author data ,55, we fependent . It is cependent ,18,57. Ptrasound .Upon seeing these differences, we selected a few noteworthy criteria to that could possibly help surgeons and pediatricians to understand how to suspect MIS-C in order to start treatment promptly. We found that if the patient suspected of acute appendicitis had three out of four criteria: CRP > 55.8 mg/L, duration of symptoms lasting three days or more, symptoms of other organ system involvement  as well as a history of a febrile fever of at least 38 degrees Celsius—he or she should be examined and tested for possible MIS-C, especially with a known recent prior COVID-19 infection, contact or a positive COVID-19 IgG test. It is important to mention that a patient with a perforated acute appendicitis might present similarly; therefore, we warrant to be cautious not to overlook this possibility and possibly still opt for surgical treatment when clear clinical, laboratory and ultrasound signs of acute appendicitis are present, as cases of solitary MIS-C appear to have less prominent abdominal symptoms as well as have a normal appendiceal diameter on ultrasound. Another possible problem with using our criteria is that gastrointestinal symptoms are usually the more prominent ones and other organ system involvement might be overlooked if the symptoms are mild and not tested for, and they sometimes tend to appear later during the progression of the disease. Fever also might not start on disease onset, but it develops in almost all cases if correct treatment is not applied, which is always insufficient in cases of MIS-C if they are only treated as acute appendicitis . From clWe have studied and compared MIS-C cases both with and without confirmed acute appendicitis as well as COVID-19 positive acute appendicitis cases and compared them to regular acute appendicitis cases. We have found that MIS-C should be suspected even with clear acute appendicitis symptoms and laboratory markers when at least three out of four signs are present: CRP > 55.8 mg, symptoms last three days or longer, presence of febrile fever and any kind of other system involvement is noticed, especially with a known prior recent COVID-19 contact, infection or positive COVID-19 IgG antibody test. We advise to test for MIS-C but stress the importance of understanding that it can also present together with complicated forms of acute appendicitis. Cases of COVID-19 positive acute appendicitis appear to be both differ from MIS-C or separate acute appendicitis cases and are in need of more investigation.


The results suggest that SARS-CoV-2 transmission can be reduced in school classes by mandatory FFP-2 mask use. In many cases, however, infection appears to be postponed rather than avoided.In this retrospective cohort study involving 614 secondary school students, the likelihood of becoming infected with SARS-CoV-2 in schools with different focus  and different prevailing restrictions were compared. A significantly higher likelihood of infection with SARS-CoV-2 was found in sports classes during the period with a strict FFP-2 mask requirement compared to general branch classes . The higher likelihood of infection was observed both during the Delta and the Omicron wave. After the relaxation of the mitigation measures, however, students in general branch classes showed a clear “catch-up” of infections, leading to a higher incidence of infections during this phase. By the end of the observation period (30 April 2022), only a small difference in cumulative SARS-CoV-2 infection rates ( Worldwide, different restrictions were mandated to control the spread of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. The stringency of these was based on the predominant viral load in the national population . As one The aim of this study was to assess if wearing face masks in the classroom is a useful mitigation measure to control SARS-CoV-2 infections and to gain an impression whether this effect is big enough to override potential negative “side effects”.In this retrospective cohort study, we compared cumulative SARS-CoV-2 infection rates in sports classes (with limited use of face masks) and non-sports classes (with the consequent use of face masks). The study was registered in the German Clinical Trial Register (ID DRKS00029061) and approved by the Research Ethics Committee at the University of Graz, Styria, Austria (GZ. 39/70/63 ex 2021/22).A school campus in Klagenfurt, Austria, was selected, consisting of a secondary school with a focus on “development for competitive sports”, and a parallel general school branch (GB). Children/adolescents attending the branch with a sports focus (SF) were allowed to participate in sports and other physical activity without restriction, due to the guidelines and safety measures for competitive sports in Austria . In SF cStudents attending the GB were allowed to carry out physical activities and sports only under strict restrictions in compliance with existing COVID-19 mitigation measures ); four-year high school (H.S.): age 14–20 years (mean 16.9 [95%CI = 16.8–17.1 years])) and sex .Descriptive statistics were calculated; continuous variables are expressed as mean (M) and standard deviation (SD), and categorial variables as absolute values (n) and percentages (%), and no data imputation was performed.2) or a Fisher’s exact test where appropriate to test for differences in cumulative percentages of students with a SARS-CoV-2 infection in SF and GB classes.We used a chi-square test (XPhi coefficients (φ) were calculated to estimate the correlation strength between stringent mitigation measures in the SF and GB school classes.Additionally, a binary logistic regression analysis was performed to assess the relationship between class membership (SF or GB) and the cumulative percentage of students with a SARS-CoV-2 infection (CPI) or the potential SARS-CoV-2 cases (infections) in classroom settings. Coefficients obtained from binary logistic regression were expressed as odds ratios (OR) with 95% confidence intervals. Due to a lack of variance homogeneity, we used the Welch test to analyze differences in seven-day incidences.p-value of <0.05 considered statistically significant. Phi (φ) according to Cohen [All tests were two-sided, with a to Cohen  was usedp ≤ 0.001). Students in SF classes were slightly younger than those in GB classes   included in this analysis, 195 students (31.8%) attended school classes with a sports focus (SF) and were allowed to participate in sports without restrictions during the study period. Significantly fewer girls were in SF classes than in GB classes  .p = 0.011). No differences in previous infections were reported by younger students   had been infected with SARS-CoV-2 before the study period (before 13 September 2021). More previous infections were reported by older students (H.S.) in SF classes than by students in parallel GB classes  .p = 0.30; and φ = 0.05) (No significant differences were found in the CPI by the end of the time period 1 (P1) between SF and GB classes, and overall the CPI levels were very low  .p = 0.008, φ = 0.11). For students in SF school classes, the odds of becoming infected with SARS-CoV-2 increased during P2 ; the end of December 2021: OR = 1.97 ). ORs were higher for younger (M.S.) students ; the end of December 2021: OR = 2.59 ) than older (H.S.) students ; the end of December 2021: OR = 1.53 ) (In time period 2 (P2), the CPI increased and moderate differences were observed ) .p < 0.001; and φ = 0.23). At the end of February 2022, students in the SF school classes exhibited a 2.6-fold increased probability of having been infected with SARS-CoV-2 )  due to the Omicron wave. By the end of February 2022, greatly increased CPI in both branches went along with a significant difference between the school focuses ) .p = 0.018, φ = 0.10). This trend continued until the end of April 2022  and could also be observed in different school grades (M.S. and H.S.)  .Detailed information on different CPI trends in subgroups are reported in the p < 0.001) between school focuses in all four time periods. In P1, P2, and P3, higher (p < 0.001) M7D-I SARS-CoV-2 was observed in SF school classes. During P4, the situation reversed, and significantly higher (p < 0.001) M7D-I SARS-CoV-2 was detected in GB school classes  had been used. Due to the limited number of participants, GT for two days during P1, P2, and P4 partially identified insufficient numbers of potential infection cases. Regardless of which GT was used, there were no significant changes in the overall conclusion of which school branch was more likely to be infected with SARS-CoV-2 .In P2 and P3, the odds of PI-SARS-CoV-2 were higher in SF classes ; P3: OR = 4.03 ). These changes in P4, where the odds of PI-SARS-CoV-2 were significantly reduced in SF classes, were in contrast to those in P2 and P3 ) .Additional results for differentially hypothesized GTs are reported in the To our knowledge, our study is the first to assess the impact of wearing face masks in classroom settings. Our results show that under otherwise strict mitigation measures, students allowed to participate in sports without restriction (SF group) were at a significantly higher risk of being infected by SARS-CoV-2 than those following general strict mitigation measures at school. This is in line with the results of studies reporting the clear efficacy of wearing FFP2 or any other face masks, with both leading to a reduction in viral (variant) spreading ,34.To the best of our knowledge, the long-term effects of continued mask wearing have been investigated in only a few studies to date ,36,37,38In late February 2022, an increasing number of studies reported that Omicron variants led to less severe disease, and reduced hospitalization rates. In response, stringent mitigation measures were relaxed worldwide ,40. Our This leads to the question when, for whom, and which face masks should be made obligatory in school settings, and especially for how long. These questions must be answered by medical research and should no longer be a matter of individual/local/regional decision-making. Although no major physical/physiological negative side effects of face mask use have been proven for healthy infants so far , continuDue to the limited number of school classes without strict mitigation measures during the school year 2021/2022, no random representative sample selection was possible. However, the sample size was large and the participation rate was high (99.7%).Another limitation of our study is that the actual chain of SARS-CoV-2 transmissions cannot be detected flawlessly in practice. In fact, transmission could also have taken place outside school by personal contacts during leisure time. This limitation, however, holds true for almost all studies dealing with secondary attack estimations.Regardless of the limitations, our results clearly show the “catch-up” of infections in response to the relaxation of strict mitigation measures. Thus, the temporary obligation of face mask use in schools can postpone a number of secondary infections but has a very limited effect on the long-term avoidance of these infections. This finding should be considered regarding further decisions concerning obligatory face mask use in school settings.Our study shows that a number of infections with SARS-CoV-2 are delayed, but they cannot be prevented in the long run by wearing face masks. Therefore, the obligatory use of face masks in schools may be understood as an epidemiological measure to flatten SARS-CoV-2 peaks rather than to protect individuals. Since healthy school children are rarely severely affected by COVID, on the one hand, but may experience negative psychosocial consequences, on the other hand, by continued face mask use, the advantage of (temporarily) reduced virus transmission must be carefully balanced against the potential negative consequences on psychosocial development and mental health.


Recently, the neurite outgrowth inhibitor-B (Nogo-B) receptor has been reported as a novel candidate gene for Parkinson's disease (PD). Nogo-B receptors need to combine with soluble Nogo-B to exert their physiological function. However, little is known about the relationship between serum soluble Nogo-B and PD.Serum levels of sNogo-B and α-Synuclein (α-Syn) were measured in a cohort of 53 patients with PD and 49 healthy controls with the ELISA kit method.p = 0.049), H&Y stage (p = 0.0108) as well as serum α-Syn level (p = 0.0001). The area under the curve (AUC) of serum sNogo-B in differentiating patients with PD from controls was 0.801 while the AUC of serum α-Syn was 0.93. Combining serum sNogo-B and α-Syn in differentiating patients with PD from HC presented higher discriminatory potential (AUC = 0.9534).Serum sNogo-B level is significantly lower in the PD group than that in healthy controls and is negatively correlated with UPDRS-III score (Decreased serum sNogo-B may be a potential biomarker for PD. Lower Nogo-B level reflects worse motor function and disease progression of PD. Serum sNogo-B is of added value to serum α-Syn panel in distinguishing PD from controls. Future studies are needed to confirm in larger samples and different populations. Parkinson's disease (PD) is one of the most common neurodegenerative disorders. It has affected over 1% of people beyond 65 years old around the world Tanner, . PD is pNowadays, there are many studies on diagnostic biomarkers of PD such as blood  receptor  and 49 age-and sex-matched healthy controls from the Department of Neurology of First Affiliated Hospital of Guangxi Medical University between August 2017 and September 2019. PD's diagnostic criteria followed the United Kingdom PD Society Brain Bank   were used to determine sNogo-B levels in the serum of patients with PD. In order to verify our determination with prior studies, the levels of α-Synuclein in serum were determined by Abcam Human α-Synuclein ELISA Kit . The samples were diluted if necessary during the assay. All of the above procedure was carried out in strict accordance with the instructions.p > 0.05). Comparisons between groups were made using the Chi-squared test for categorical data, and independent samples t-test or the Mann–Whitney U-test for continuous variables. A receiver operating characteristic curve was drawn to calculate the area under the curve for sensitivity and specificity. Pearson's or Spearman's rank correlation analyses were conducted to evaluate the correlation between levels of sNogo-B, α-Synuclein, and clinical variables.All statistical analyses were performed with IBM SPSS Statistics 23 and all data were tested for normality of distribution (p < 0.0001) while serum α-Syn levels were observed higher in PD group than that in HC group , as illustrated in p = 0.521) as well as α-Syn levels , which was shown in Patients with PD and healthy controls were matched in age and gender. Serum sNogo-B levels were observed lower in PD group than that in HC group  of serum sNogo-B in differentiating patients with PD from HC was 0.801 and the cutoff value was 209.09 pg/ml; Sensitivity and specificity were 91.1 and 49%, respectively. While AUC of serum α-Syn in differentiating patients with PD from HC was 0.93 and the cutoff value was 3,712.67 pg/ml. Sensitivity and specificity were 86.8 and 85.7%, respectively. The AUC of combining serum sNogo-B and α-Syn in differentiating patients with PD from HC was 0.9534 and sensitivity and specificity were 94.3 and 85.7%, respectively .r = −0.264, p = 0.049) and negatively correlated with the Hoehn and Yahr stage  and serum α-Syn level , respectively  as well as levodopa equivalent dose .Pearson or Spearman correlation analysis showed that serum sNogo-B level was mildly negatively correlated with UPDRS III scores , Guangxi Natural Science Foundation Program (No. 2019GXNSFDA185008), and Guangxi Medical and Health Appropriate Technology Development and Application Project (No. S2019095).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PX declared a shared affiliation with the author WG to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Paclitaxel is widely used in the treatment of various types of solid malignancies. Paclitaxel-induced peripheral neuropathy (PIPN) is often characterized by burning pain, cold, and mechanical allodynia in patients. Currently, specific pharmacological treatments against PIPN are lacking. Curcumin, a polyphenol of Curcuma longa, shows antioxidant, anti-inflammatory, and neuroprotective effects and has recently shown efficacy in the mitigation of various peripheral neuropathies. Here, we tested, for the first time, the therapeutic effect of 1.5% dietary curcumin and Meriva (a lecithin formulation of curcumin) in preventing the development of PIPN in C57BL/6J mice. Curcumin or Meriva treatment was initiated one week before injection of paclitaxel and continued throughout the study (21 days). Mechanical and cold sensitivity as well as locomotion/motivation were tested by the von Frey, acetone, and wheel-running tests, respectively. Additionally, sensory-nerve-action-potential (SNAP) amplitude by caudal-nerve electrical stimulation, electronic microscopy of the sciatic nerve, and inflammatory-protein quantification in DRG and the spinal cord were measured. Interestingly, a higher concentration of curcumin was observed in the spinal cord with the Meriva diet than the curcumin diet. Our results showed that paclitaxel-induced mechanical hypersensitivity was partially prevented by the curcumin diet but completely prevented by Meriva. Both the urcumin diet and the Meriva diet completely prevented cold hypersensitivity, the reduction in SNAP amplitude and reduced mitochondrial pathology in sciatic nerves observed in paclitaxel-treated mice. Paclitaxel-induced inflammation in the spinal cord was also prevented by the Meriva diet. In addition, an increase in α7 nAChRs mRNA, known for its anti-inflammatory effects, was also observed in the spinal cord with the Meriva diet in paclitaxel-treated mice. The use of the α7 nAChR antagonist and α7 nAChR KO mice showed, for the first time in vivo, that the anti-inflammatory effects of curcumin in peripheral neuropathy were mediated by these receptors. The results presented in this study represent an important advance in the understanding of the mechanism of action of curcumin in vivo. Taken together, our results show the therapeutic potential of curcumin in preventing the development of PIPN and further confirms the role of α7 nAChRs in the anti-inflammatory effects of curcumin. Chemotherapeutic agents used in cancer therapy can cause peripheral neuropathy due to their neurotoxicity . It is eThe symptoms described by patients are chronic pain, loss of sensation and allodynia in the feet and hands according to a “stocking and glove” distribution, as all of which significantly impact their quality of life . CurrentCurcuma longa, has been shown to improve functional recovery in many animal models of peripheral neuropathy ). Post-hoc analysis showed no difference in mechanical sensitivity between the three groups of vehicles at the different time points (BL to D21). However, a significant decrease in the paw-withdrawal threshold was observed in the paclitaxel-treated animals at D7 (p < 0.001), D14 (p < 0.001) and D21 (p < 0.001), reflecting paclitaxel-induced mechanical hypersensitivity. Treatment with the curcumin diet at 1.5% partially prevented this hypersensitivity at D14 and D21. Whereas, treatment with the Meriva diet at 1.5% completely prevented the mechanical hypersensitivity induced by paclitaxel to D14 and partially to D7 and D21  C. An ove and D21 C. These p < 0.0001]). More specifically, no difference was observed between the three vehicle groups. However, a significant increase in the time aversive response was observed at D7 (p < 0.05), D14 (p < 0.05), and D21 (p < 0.01) between reg-diet/veh and reg-diet/PAC groups, reflecting that cold hypersensitivity was induced by paclitaxel  in the paclitaxel-treated group (p < 0.001). No significant difference was observed between the control group and the cur-diet/PAC and Meriva-diet/PAC groups (p < 0.001) and Meriva-diet/PAC (p < 0.05) groups and the paclitaxel-treated group (reg-diet/PAC); Finally, sensory-nerve-conduction amplitude E and velC groups F. In addOne of the characteristics of paclitaxel neurotoxicity is the presence of mitochondrial damage in the axons of the peripheral nerves. Thus, a qualitative analysis by electron microscopy was performed on the longitudinal sections of the sciatic nerve of the animals, in order to characterize their mitochondrial morphology . Under cFor the paclitaxel-treated group of animals (reg-diet/PAC), there was clear evidence of mitochondrial pathology, as previously described in the literature . We obseTreatment with the 1.5% curcumin and 1.5% Meriva diets reduced the mitochondrial pathology induced by paclitaxel . In bothp < 0.05; p < 0.05)  A. No sigAfter comparing the two curcumin preparations in the various aspects of PIPN, we decided to further study the mechanisms of prevention by the Meriva diet, which showed enhanced bioavailability and better results on PIPN signs in the von Frey and wheel-running behavioral tests.An analysis of the levels of the primary inflammatory markers was performed in the DRG and lumbar spinal cord in the multiplex assay at Day 22 after the behavioral testing .p < 0.05).As shown in In the lumbar spinal cord B, we obsTaken together, these results show that on Day 22, paclitaxel-induced inflammation occurred primarily in the lumbar spinal cord and that Meriva-diet reduces this neuroinflammation.p < 0.01)  , the lev < 0.01) B. No cha < 0.01) B.Based on the results obtained for the spinal increase in α7 nAChRs expression, an intrathecal injection of an α7 nAChR antagonist (MLA) was performed to determine if the effects of Meriva previously observed with the von Frey test were dependent on the spinal α7 nAChRs. Our results show that 14 days after the first injection of paclitaxel, the Meriva diet partially reversed the mechanical hypersensitivity induced by PAC. Interestingly, the injection of MLA at D15 leads to a decrease in the withdrawal threshold in the Meriva-diet/PAC group compared to the previous day without MLA injection. These findings suggest that the spinal α7 nAChRs mediate the beneficial effects of Meriva in the development of paclitaxel-induced mechanical hypersensitivity.The beneficial effect of Meriva in PIPN is largely dependent on α7 nAChRs.Based on the previous results showing that the α7 nAChR antagonist blocks the effects of Meriva-diet treatment on mechanical hypersensitivity, and a more detailed study was conducted using α7 nAChR global KO mice. Behavioral and electrophysiological measurements were conducted on α7 nAChR KO mice treated with paclitaxel and supplemented with the Meriva diet over 14 days .p < 0.05). However, at D7 and D14, no significant difference was found between the KO and WT vehicle groups (WT reg-diet/Veh and KO reg-diet/Veh). The Meriva diet did not induce any difference in mechanical sensitivity in the KO and WT vehicle groups (WT Meriva-diet/Veh and KO Meriva-diet/Veh) at D7 and D14. The KO and WT mouse groups treated with paclitaxel and the regular diet showed significantly lower paw-withdrawal thresholds than the controls at D7  and D14 , which is indicative of paclitaxel-induced mechanical hypersensitivity in these two groups. As before, the Meriva diet significantly decreased the development of paclitaxel-induced mechanical hypersensitivity in the WT group, compared with the regular diet paclitaxel-treated group at D7 (p < 0.05) and D14 (p < 0.001), but did not completely block it (D7: p < 0.05 and D14: p < 0.001 vs WT reg-diet/Veh). However, consistent with the involvement of the α7 nAChRs in paclitaxel-induced toxicity, the preventive effect of the Meriva-diet on paclitaxel-induced mechanical hypersensitivity was largely diminished in the KO mouse group. Thus, these results suggest that the effect of Meriva on mechanical hypersensitivity is dependent on α7 nAChRs.Our data from the von Frey test A show thp < 0.001; D14: WT Meriva-diet/PAC vs WT reg-diet/PAC p < 0.001). However, the preventive effect of the Meriva diet was not evident in the PAC KO group , again suggesting a critical role for α7 nAChRs in the preventive effect of curcumin.Cold-sensitivity results B show thp < 0.05). This decrease in voluntary wheel-running activity was prevented by the Meriva-diet (WT Meriva-diet/PAC vs WT reg-diet/PAC: p < 0.01). A trend towards a decrease in voluntary wheel-running activity was also observed in KO animals treated with paclitaxel (p = 0.07). The same trend was observed in KO animals treated with paclitaxel and the Meriva-diet (p = 0.07).On day 7, voluntary wheel-running activity C was sigp < 0.05) that was prevented by the Meriva diet (WT Meriva-diet/PAC vs WT reg-diet/PAC: p < 0.05). A significant decrease in SNCAP amplitude was also observed in the KO group treated with paclitaxel (KO reg-diet/Veh vs KO reg-diet/PAC: p < 0.05). However, the Meriva diet did not prevent this decrease in amplitude in KO mice.Concerning the electrophysiological data measured at D21 D, a signTaken together, these results demonstrate that the effect of Meriva and, thus, curcumin in PIPN is largely dependent on α7 nAChRs.The anti-inflammatory effects of the Meriva diet observed in paclitaxel-treated WT mice  were tesp < 0.01), IL-3 (p < 0. 001), IL-4 (p < 0.05), IL-12(p40) (p < 0.001), IL-17α (p < 0.01), G-CSF (p < 0.01), GM-CSF (p < 0.001), KC (p < 0.001), MIP-1b (p < 0.05) and a trend for TNF-α (p = 0.07) in both regular and Meriva-diet-treated α7 nAChR KO mice compared to the control (WT mice). A significant reduction in MIP-1α (p < 0.001) and RANTES (p < 0.05) was also observed  and is known for its antioxidant, anti-inflammatory, and antibacterial effects. Over the last 50 years, it has been shown that the beneficial effects of tumeric powder are derived from curcuminoids, and in particular, from curcumin [The root of curcumin . It has curcumin ,35. Varicurcumin . In the curcumin ,37. Howecurcumin ,24. Our curcumin . The lowIt has previously been shown that curcumin can improve the signs of peripheral neuropathy in models of traumatic, metabolic, and genetic injury . This ha2+, could modulate the excitatory and inhibitory component of synaptic transmission in peripheral neurons, due to their presence on glutamatergic and GABAergic nerve terminals [Our results suggest that the α7 nAChR subtype may mediate, to a large extent, the protective effects of curcumin in the mouse PIPN model. Meriva-diet increased expression of this nicotinic subunit in the spinal cord but not in the DRGs in paclitaxel-treated mice only. Furthermore, injection of a selective α7 nAChR receptor antagonist intrathecally completely blocked the partial protective effect of Meriva on the development of paclitaxel-induced mechanical hypersensitivity. These results were confirmed by using α7 nAChR KO mice, in which the protective effect of Meriva on the development of mechanical and cold hypersensitivity, as well as the decrease in the SNCAP amplitude, was blocked. In addition, the decrease in neuroinflammation in the spinal cord by Meriva that was observed in WT mice was not observed in α7 nAChR KO mice. These results extend our previous data on the role of α7 nAChRs in the beneficial effect of curcumin in acute-inflammatory-mouse models and with curcumin as a PAM of expressed α7 nAChRs . It is kerminals ,48. Thuserminals . Furthererminals ,50.In addition to its potential for mitigation of CIPN, numerous studies suggest that curcumin represents a promising candidate as an effective anticancer drug to be used alone or in combination with other drugs . HoweverSeveral clinical trials have already been conducted in humans in chronic-inflammation and joint-pain pathologies using different formulations of curcumin ,52,53. FIn conclusion, this study shows for the first time that curcumin can largely prevent the development of PIPN in rodents. Thus, in this study, significant improvements were observed with curcumin treatment at the histological, electrophysiological, and functional levels. Moreover, for the first time, we show in vivo that the anti-inflammatory and antinociceptive effects of curcumin in peripheral neuropathy are dependent on α7 nAChR receptors in spinal cord. Curcumin, having already been tested in humans in various clinical trials and having no known toxicity, should be the subject of a new study in the treatment of peripheral neuropathies. This study confirms once again the therapeutic potential of novel formulations of curcumin in the treatment and prevention of peripheral neuropathies.


Staphylococcus aureus ATCC 25923 were analyzed by the bacteriostatic activity test. Differences in intracellular metabolites of S. aureus following 19 μg/ml BBR exposure for 1 h were investigated by combining non-targeted metabolomics techniques of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The results showed that the minimum inhibitory concentration of BBR against S. aureus was 51 μg/ml. A total of 368 and 3,454 putative metabolites were identified by GC-MS and LC-MS analyses, respectively. Principal component analysis showed the separation of intracellular metabolite profiles between BBR-exposed samples and non-exposed controls. Pathway activity profiling analysis indicated a global inhibition of metabolisms by BBR exposure, while enhancement was also found in nucleic acid metabolism, amino sugar, and nucleotide sugar metabolism. Several metabolic markers were screened out mainly based on their variable importance of projection values. Two pyridine dicarboxylic acids were significantly downregulated, suggesting the reduction of stress resistance. The oxidized phospholipid (PHOOA-PE) was accumulated, while lipid antioxidant gamma-tocopherol was decreased, and farnesyl PP, the synthetic precursor of another antioxidant (staphyloxanthin), was decreased below the detection threshold. This evidence indicates that BBR reduced the antioxidant capacity of S. aureus. Accumulation of the precursors  and downregulation of the key metabolite D-Ala-D-Ala suggest the inhibition of cell wall synthesis, especially the peptidoglycan synthesis. Metabolites involved in the shikimate pathway (such as 3-dehydroshikimate) and downstream aromatic amino acid synthesis were disturbed. This study provides the first metabolomics information on the antibacterial mechanism of BBR against S. aureus. The key metabolic markers screened in this study suggest that the shikimate pathway, staphyloxanthin synthesis, and peptidoglycan biosynthesis are new directions for further study of BBR antibacterial mechanism in the future.Berberine hydrochloride (BBR) is a natural product widely used in clinical medicine and animal production. It has a variety of antimicrobial effects, but its complex antimicrobial mechanism has not been clarified. This study aimed to discover the metabolic markers and gain a new perspective on the antibacterial mechanism of BBR. The effects of different inhibitory concentrations of BBR on the survival and growth of standard strain  Staphylococcus aureus is a common bacterial colonizer of human and a variety of animal species which can cause many infectious diseases, such as mastitis, endocarditis, septic arthritis, and sepsis. In the treatment of infectious diseases, antibiotics were initially highly effective, but they currently show several limitations, including widespread resistance, toxicity, and residues. S. aureus has developed resistance to various antibiotics including vancomycin , fungi, parasites, and even viruses  and play a synergistic antimicrobial role with clindamycin or rifamycin  in many medicinal plants, such as S. aureus through extensive screening of metabolic markers and analysis of related metabolic pathways.Analyzing the expression changes of specific genes in microorganisms is a common method to explain the antimicrobial effect of berberine. The intervention on gene expression level (including transcription and protein synthesis) may be indirect, so it is difficult to explain the antimicrobial mechanism of berberine. Metabolites are the final products of gene expression, and the metabolic alterations represent the exact physiological state of microorganisms . Before use, the strain was subcultured onto nutrient agar plates for 24 h at 37°C. Then, the single colony was selected and coated on a nutritious agar plate to grow for 7 h, and the culture would be used as a “seed” to cultivate working bacteria culture (WBC) for subsequent experiments. For the preparation of WBC, the “seed” was inoculated into the broth medium  with an initial inoculation dose of 107 CFU/ml and then cultured with shaking at 37°C and 120 rpm for ~2.5 h to enter the logarithmic growth phase (OD600 ~ 0.3).The BBR (HPLC ≥ 98%) and vancomycin (potency ≥ 900 μg/mg) were purchased from Desite Bio-Tech Co., Ltd.  and Solarbio Bio-Tech Co., Ltd. , respectively, and were dissolved in sterilized ultra-pure water before use. The reagents used in the preparation of culture medium were purchased from Aoboxing Bio-Tech Co., Ltd. . The standard strain S. aureus (~108 CFU/ml) at 37°C. The lowest BBR concentration without visible color change and turbidity change within 24 h was MIC. The bacterial cultures exposed to different concentrations of berberine for 24 h were coated on nutrient agar plates and cultured at 37°C for 24 h. The MBC was determined by the lowest berberine concentration without bacterial growth.The MIC value was determined by the improved broth dilution method to improve the test sensitivity  was diluted with the same volume of broth medium containing BBR to give an approximate starting inoculum of 108 CFU/ml and to obtain final BBR concentrations of 1/4 × MIC, 3/8 × MIC, 1/2 × MIC, or MIC. The control culture was added with the same volume of broth medium without BBR. The cultures were incubated further for 8 h, and cell growth was monitored spectrophotometrically . Time kill tests of the above BBR concentrations were assessed by counting CFU  and exposure time (1 h) for the treatment of metabolic samples were determined by the result of time kill tests. The culture conditions were the same as the antibacterial effect test. Therefore, metabolic samples (group T1) were collected at the 1-h time point after being exposed to 19 μg/ml BBR. Two control groups (without BBR addition) collected at zero time point were treated as the initial control group (C0) and those collected 1 h later were treated as the growth control group (C1).Intracellular metabolites were extracted using cold methanol and chloroform  was utilized to separate the derivative metabolites. For LC-MS analysis, the freeze-dried samples were re-extracted and analyzed by a Nexera UPLC system coupled with Q Exactive quadrupole-orbitrap mass spectrometer equipped with heated electrospray ionization (ESI) source. An ACQUITY UPLC HSS T3 column  was employed in both positive and negative modes. Metabolome analysis was performed by Lu-Ming Biotech Co. Ltd.  [see details in the supporting information ].The MS-DIAL software was used to preprocess GC-MS data. Metabolite characterization is based on the LUG database (a self-built untarget database of GC-MS from Lu-Ming Bio). All peak signal intensities in each sample were segmented and normalized according to the internal standards with RSD > 0.3 after screening. LC-MS data were processed by software Progenesis QI V2.3. Main parameters of 5 ppm precursor tolerance, 10 ppm product tolerance, and 5% production threshold were applied. Compound identification was based on the precise mass-to-charge ratio (m/z), secondary fragments, and isotopic distribution using HMDB, Lipidmaps (V2.3), Metlin, EMDB, PMDB, and self-built databases to do qualitative analysis. Peaks with a missing value (ion intensity = 0) in more than 50% of groups were removed. Zero value was replaced by half of the minimum value. Compounds with resulting scores below 36 (out of 60) points were deemed to be inaccurate and removed.T-test was further used to verify whether the metabolites of difference between the two groups were significant. P-value adjustment was performed using the Benjamini-Hochberg method . Heatmaps  were plotted by http://www.bioinformatics.com.cn, a free online platform for data analysis and visualization. Metabolomics datasets and metabolic pathway activities were correlated using the pathway activity profiling (PAPi) algorithm  were analyzed by the PAPi_1.8.0 package in R.The data matrix of the metabolome was imported in R to carry out principle component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA). To prevent overfitting, 7-fold cross-validation and 200 response permutation testing (RPT) were carried out to evaluate the quality of the OPLS-DA models. Variable importance of projection (VIP) values obtained from the OPLS-DA model were used to rank the overall contribution of each variable to group discrimination. A two-tailed Student's m Aggio, . Only thS. aureus strain ATCC 25923 was 51 μg/ml, and the MBC value was two times the value of MIC (102 μg/ml). When combined with vancomycin, BBR of 1/2 × MIC (25.5 μg/ml) halved the MIC value (2 μg/ml) of vancomycin against S. aureus strain ATCC 25923 ]. Six other significant metabolites involved in peptidoglycan biosynthesis were identified (2FC = −2.4 (−3.6)] restricts the upstream part of peptidoglycan biosynthesis pathway. Farnesyl diphosphate (farnesyl-PP), the precursor metabolite of undecaprenyl pyrophosphate (Und-PP) in the peptidoglycan biosynthesis pathway, was found to be decreased below the detection threshold in the T1 group, while it was detected in all the 12 samples of the control groups [log2FC = −29.5 (−29.3)]. L-alanine [log2FC = −1.4 (−2.7)] and glycine [log2FC = −1.9 (−2.0)] were downregulated, while L-glutamine was accumulated [log2FC = 2.1 (3.4)]. The precursors of teichoic acid biosynthesis  were also accumulated significantly ] and 2,6-pyridinedicarboxylic acid [log2FC = −8.3 (−9.0)] were significantly decreased after BBR treatment. They belong to pyridine dicarboxylic acid, which is known to be abundant in bacterial spores and endows spores with stress resistance ], which is known as a lipid vitamin and acts as an antioxidant . The oxidized glycerol phospholipid (PHOOA-PE) was not detected in the control groups, but was accumulated in the T1 group (log2FC = 32.5).The VIP-top five metabolites in GC-MS dataset were desyrel, 2,5-pyridinedicarboxylic acid, 2,6-pyridinedicarboxylic acid, gamma-tocopherol, and 3-dehydroshikimate . 2,5-pyr Setlow, . Gamma-t2FC = −5.9 (−6.9)] and chorismate [log2FC = 6.3 (7.2)], showed opposite regulatory trends. L-phenylalanine, L-tyrosine, and L-tryptophan, which were synthesized using chorismate as precursors, were downregulated  was accumulated , indicatS. aureus, such as the reduction of gamma-tocopherol and farnesyl-PP, and the accumulation of PHOOA-PE   and the Chengdu University of TCM Science Foundation (BSH2019010). The authors declare that this study received funding from Science and Technology Department of Sichuan Province and Chengdu University of TCM. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.JN was employed by Guangdong Meilikang Bio-Sciences Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Freezing of gait (FOG) is a particularly debilitating symptom of Parkinson’s disease (PD) and is often refractory to treatment. A striking feature of FOG is that external sensory cues can be used to overcome freezing and improve gait. Local field potentials (LFPs) recorded from the subthalamic nucleus (STN) and globus pallidus (GP) show that beta-band power modulates with gait phase. In the STN, beta-band oscillations are modulated by external cues, but it is unknown if this relationship holds in the globus pallidus (GP). Here we report LFP data recorded from the left GP, using a Medtronic PC + S device, in a 68-year-old man with PD and FOG during treadmill walking. A “stepping stone” task was used during which stepping was cued using visual targets of constant color or targets that unpredictably changed color, requiring a step length adjustment. Gait performance was quantified using measures of treadmill ground reaction forces and center of pressure and body kinematics from video monitoring. Beta-band power (12–30 Hz) and number of freezing episodes were measured. Cues which unpredictably changed color improved FOG more than conventional cues and were associated with greater modulation of beta-band power in phase with gait. This preliminary finding suggests that cueing-induced improvement of FOG may relate to beta-band modulation. Gait disorder is one of the most disabling motor symptoms of Parkinson’s disease (PD), particularly the phenomenon of freezing of gait (FOG). FOG is defined as a “brief, episodic absence or marked reduction of forward progression of the feet despite the intention to walk”  and is oRecent findings from studies that recorded local field potentials (LFPs) from implanted deep brain stimulation (DBS) leads showed that neural activity in the basal ganglia modulates with gait phase . MoreoveA 68-year-old man who had been diagnosed with PD for 11 years was unilaterally implanted with a Medtronic 3389 electrode in the left GP and a Medtronic Activa PC + S sensing pulse generator . His OFFThe participant was tested in the morning after 12-h withdrawal from antiparkinsonian medications  and DBS ON. He performed the walking task on an instrumented treadmill . Stimulation was applied at an amplitude of 3.5 V, a frequency of 140 Hz and a pulse width of 60 μs. LFP data from the PC + S was synchronized with center of pressure (COP) data from the treadmill in the following manner. Briefly turning off/on DBS provided a synchronization signal that was present on the LFP channel and could be detected from a surface EMG sensor  on the participant’s neck. Onset and offset of treadmill movement were recorded with an external tachometer  connected in synch to the same A/D converter  as the EMG sensor. The onset/offset of treadmill movement was also recorded in another tachometer internal to the treadmill. The signal from the internal tachometer and COP data, sampled at 500 Hz, were exported from the treadmill system. This procedure allowed us to synchronize LFPs with EMG, EMG with treadmill movement, and treadmill movement with COP, from which gait phase was determined as described below. Video of the lower extremities was recorded at 60 Hz with a spatial resolution of 720p .3 isotropic) and a high resolution T2-weighted axial slab covering from the top of the thalamus to the base of the substantia nigra with (0.4 × 0.4 × 1.0 mm3) voxel size. The globus pallidus internus (GPi) and globus pallidus externus (GPe) were manually segmented on the 7 T T2 axial image following out previous protocol (https://www.slicer.org/). The T2-weighted image was linearly co-registered to the patient’s T1-image using the Advanced Normalization Tools , equipped with SC72 gradients capable of 70 mT/m and a 200 T/m/s slew rate using a 32-element head array coil . Dielectric pads were utilized to enhance signal in the temporal regions . The scaprotocol . The GPion Tools . Postopeocations  were detocations .The raw time domain LFP signal was downloaded from the PC + S device and processed offline in Python. Using custom Python code, the LFP data was band-pass filtered between 12 and 30 Hz, squared, moving average filtered (with window size of 0.1 s), and square-rooted to get the envelope of beta-band RMS   to matchA linear mixed effects model with 12–30 Hz beta power values as dependent variable was constructed to examine the effect of cueing on the LFP. Condition (color changing vs. non-color changing cues), Gait phase , and their interaction (Condition by Gait phase) were fixed factors with Trial as the random-intercept term. In order to extract the beta power for each phase, we defined each phase using the average gait phase value  with events of bilateral heel-strikes and toe-offs exported from the instrumented treadmill. Then, we used the LFP sampling frequency and the average duration of a full gait cycle to estimate the number of data points in each phase. In each trial, average power value was computed for each gait phase; then these average power values were entered into the mixed model.W = 0.85, p < 0.01), a non-parametric Wilcoxon signed rank paired sample test was used to examine the difference in freezing episodes between the trials with vs. without color-changing cues. Step length was computed from heel-strike and toe-off events exported by the treadmill software, which estimated them from the COP trajectory, independently of the COP gait phase analysis. These gait events were also used to validate the COP-determined gait phase. Step lengths were normally distributed  so a paired t-test was used to examine the differences in step length between the two cueing conditions, each side analyzed separately, with Bonferroni correction. The significance level was set at 0.05. All statistical analyses were performed in R . Since the count was non-normally distributed  was near the border of GPi and GPe with the other contacts (contacts 0–2) located within GPi .The gait phase estimation based on COP data was reliable; heel-strike and toe-off events, as independently detected by the built-in treadmill software, occurred at consistent gait phases . The aveF = 31.15, p < 0.001) and Condition by Gait phase interaction  = 24.30, p < 0.001) effects. The significant interaction effect reflected that the differences between color-changing cues and constant color cues were different between swing phase and stance phase (t-tests showed that the 12–30 Hz power in color changing cues was lower during swing phase (t(32) = 3.73, p < 0.001) and greater during stance phase (t(32) = −3.24, p < 0.01) compared to constant color cues. The Condition was not statistically significant  = 0.12, p = 0.73).ce phase . Post-hoW = 72, p < 0.01, t(8) = −0.92, p = 0.76; left: t(8) = −0.60, p = 1.00).During treadmill walking, there were significantly fewer freezing episodes  the magnitude of beta-band oscillations modulated with phase of gait, 2) visual cues reduced freezing and facilitated gait  but color-changing cues, requiring a fast adjustment of stepping, improved FOG more than constant color cues, 3) beta-band modulation was greater with the color-changing cues compared to constant color cues.Previous studies with STN LFP recordings have shown beta modulation with gait phase , but thiWhy did unpredictable changes in the color of the stepping stone, which required adjustment of the ongoing gait cycle, improve FOG more than ordinary cues? It has been proposed that gait disturbances arise, in part, from a loss of automaticity and an increased reliance on attention to maintain the gait cycle . The chaIt is possible that the difference in modulation of beta-band power between the two cueing types might be a result, rather than the cause, of the decreased festination  which is strongly related to freezing . We cannSupplementary material


COVID-19 can be associated with a variety of longer-lasting impairments that can have a significant impact on patients' quality of life (QoL). While this is well described in the literature for limitations in lung capacity or permanent headaches, there is little research on the impact of olfactory dysfunction in the context of COVID-19 on patients' QoL.In 65 patients with a history of COVID-19, the present olfactory ability was assessed using the Sniffin' Sticks test. In addition, olfactory QoL was assessed by the Questionnaire of Olfactory Disorders. Self-assessment was performed with visual analogue scales. The data were compared with the results obtained in healthy individuals and in patients with hyposmia due to other viral infections.The QoL of COVID-19 patients was significantly lower compared to the healthy control group. Even recovered subjects whose olfaction had already returned to the normal range still had a reduced QoL. The severity of the olfactory impairment correlated with the reduction in QoL. However, the olfactory QoL of COVID-19 patients was not worse than that of patients' olfactory loss due to other viral infections. Patients with parosmia had reduced QoL and rated their situation worse than patients without parosmia.QoL appears to be impaired in patients with long-lasting COVID-19 olfactory disorders several months after overcoming acute symptoms, even if olfaction has normalized. However, the impairment is not more pronounced than in patients with other postviral olfactory disorders of the same duration. Olfactory dysfunction is a common symptom of COVID-19 , 3. WhilAcquired olfactory disorders can be associated with a subjective reduction in quality of life (QoL), as recently confirmed in a large multicenter study . The cauThis study was conducted at a university hospital in Germany. The principles of the World Medical Association Declaration of Helsinki and its subsequent amendments were followed and adherence was monitored by the Local Ethics Committee. Patients were recruited from the olfactory outpatient clinic for COVID-19 recovered patients and gave their written informed consent to participate in the study. The disease was initially confirmed by polymerase chain reaction and there was no need for hospitalization of any of the participants during the course of the disease. All patients had been recovering from the acute disease for at least 2 months and had reached the age of 18. Preexisting nasal diseases such as chronic sinusitis, acute allergies, trauma to the nose, or known olfactory and gustatory disorders prior to the disease were denied by all subjects. Structured questionnaires were used to collect this information, as well as other sociodemographic data such as age and gender.The data of the control groups were taken from an existing database. The controls were 32 healthy volunteers with normal olfactory function and 14 hyposmic patients who had previously been treated for postviral (not COVID associated) olfactory disorders. Orthonasal olfactory ability of the patients was measured using the felt-tip pen-based Sniffin' Sticks Test . The test allows to determine the olfactory threshold, the ability to distinguish odors, and the ability to identify odors separately. The results are presented as a composite TDI score, where values below 16.5 points correspond to functional anosmia, values between 16.5 and 30.5 points to hyposmia, and values above 30.5 points to normosmia .The subjects' QoL was assessed using the German version of the Questionnaire of Olfactory Disorders (QOD), a widely used and well-validated test of olfactory-related QoL . This teT test for independent samples. The significance of the differences between the genders of the respective groups was determined with the χ2 test. The correlations were calculated according to Spearman-Rho, the two-sided significance of the coefficients of correlation is presented.Statistical analysis was performed using SPSS v. 24 . Data are presented as means (± standard errors of the means). The mean values were compared after Levene's test for equality of variance via a n = 24, 37%) than women  (p = 0.035). The healthy control group  consisted of 19 women and 13 men  with an average age of 51.8 years (±15.0 years). The control group of patients with hyposmia due to other viral non-COVID-associated infections of the upper respiratory tract  consisted of 2 men and 12 women  with an average age of 58.6 years (±11.32 years).Sixty-five post-COVID patients were included in the study with a mean age of 44.9 years (±11.7 years). The group consisted of significantly less men (p = 0.78) and between the post-COVID patients and the postviral controls (p = 0.59). There was also no significant difference in gender distribution between the control groups (p = 0.053). In age, the post-COVID patients differed significantly from the healthy controls (p = 0.015) and from the postviral controls (p = 0.001). There was no significant age difference between the control groups (p = 0.13).There was no significant difference in gender distribution between the post-COVID patients and the healthy controls (p = 0.30).Since the acute infection, 7.1 months had passed in the COVID patients and 6.4 months in the postviral controls. This difference was not significant (The mean TDI of the post-COVID patients was 32.9 (±0.9). Fifty-three (82%) of the participants had TDI scores in the normosmic range and 10 (15%) were hyposmic. Because only 2 (3%) of the participants scored in the functionally anosmic range, these were included in the hyposmic group for calculations (hereafter referred to collectively as “hyposmic post-COVID patients” for simplicity). This was also based on the idea that there is a continuum in olfactory function between hyposmia and functional anosmia. The mean TDI within this group was 21.98 (±2.77) (Table p = 0.49), but to the hyposmic post-COVID patients (p = 0.001)   Table . The dif p = 0.00 Fig. 1)p = 0.49)p = 0.059) and healthy controls  (Table p = 0.034)  , almost double than that of the normosmic post-COVID patients , there was no such difference (p = 0.096) between the hyposmic post-COVID patients and the healthy controls   for all post-COVID patients. Within the post-COVID patients, the normosmics had a score of 2.36 (±0.46) and the hyposmics had a score of 3.13 (±0.93)   and the hyposmic post-COVID patients (p = 0.014) had significantly higher scores than the healthy controls. There was also no significant difference in QOD-P between the hyposmic post-COVID patients and the postviral controls (p = 0.13) , with normosmic post-COVID patients scoring 9.25 (±1.49) and hyposmic post-COVID patients scoring 20.75 (±4.57) (Table p = 0.96)  as well as between the post-COVID patients and the postviral controls (p = 0.001) were significant, whereas there was no significant difference between healthy controls and postviral controls (p = 0.69).In the QOD-DS, post-COVID patients scored 8.48 points (±0.33), with normosmic post-COVID patients scoring 8.47 (±0.39) and hyposmic post-COVID patients scoring 8.5 (±0.46) Table . The dif96) Fig. . The hea96) Fig. . The difr = −0.35, p = 0.020) as well as in the self-assessment visual analogue scale . QOD-QOL  and QOD-VAS  also correlated with the scores in the QOD-P.Within the group of post-COVID patients, there was a negative correlation between the TDI score and the scores in the QOD-QOL , as olfactory ability decreases with age [There were no indications that the data could be unreliable, as the post-COVID patients even had a lower score in the QOD-DS questions than the control groups. The post-COVID patients thus continue to have a significant impairment of their QoL, even if the olfactory ability is back in the normosmic range. One reason for this could be the significantly younger age of the normosmic post-COVID patients compared to the healthy control group .All the authors declare no competing interests.There is no funding for this study.Dr. M.S. Otte and Prof. J.C. Luers initiated the study. Dr. M.S. Otte and Ms. M.-L. Bork recruited and examined the patients. Prof. J.P. Klussmann, as clinic director, provided the financial resources. Prof. T. Hummel wrote and revised the paper with Dr. M.S. Otte. Prof. T. Hummel and Prof. A. Hähner provided the data of the control groups from a previously published dataset.The data of the study are with the corresponding author. The subjects have consented to anonymized processing of the data and publication, but renewed consent must be requested for data disclosure.